This invention pertains generally to the field of laser scanning microscopes and to laser scanning florescence microscopy.
Laser scanning microscopes and particularly confocal microscopes are commonly used in research for the imaging of structures such as cells. In such scanning microscopes, the light from the laser source is focused to a point within the specimen by the microscope objective, and the specimen and beam are moved relative to one another, most commonly by deflecting the light beam so that it scans across a stationary specimen. The light from the specimen is collected by the objective and passed through the microscope to the detector, such as a photomultiplier tube. Various scanning systems have been developed to deflect the beam from the source to scan across the specimen, including pairs of galvanometer driven mirrors which provide both X and Y deflections of the beam. Because such mechanically driven mirrors provide relatively slow scanning of the beam, scanning systems have been developed which provide the deflections of the beam in at least one direction by faster devices, particularly acousto-optical deflectors (AODs). A confocal laser scanning microscope having fast scanning capability which incorporates an acousto-optical deflector is shown in U.S. Pat. No. 4,863,226 to Houpt, et. al.
Scanning microscopes can also be utilized to detect fluorescence induced by the illuminating light beam, which may be carried out concurrently with the detection of the light reflected from the specimen. In conventional fluorescence microscopy, the fluorophores incorporated in the specimen are selected to absorb the illumination light at a relatively short wavelength and to fluorescently emit photons at a longer wavelength. These fluorescent photons are passed back through the scanning optics to a dichroic mirror which separates the fluorescent light from reflected light and directs the fluorescent light to a detector such as a photomultiplier tube.
Various fluorophores can absorb two or more photons of relatively long wavelengths simultaneously when sufficiently intense illumination light is applied to them, and will fluorescently emit a photon at a shorter wavelength than the wavelength of the incident light. In two photon laser scanning microscopes an incident beam of relatively long wavelength light is provided in short pulses (typically in the range of a few picoseconds to a few hundred femptoseconds per pulse) from a laser. The pulsed beam from the laser is focused onto a specimen so that the light reaches an intensity at the focal point sufficient to excite detectable two photon fluorescence. The emitted fluorescent photons are collected by the objective lens of the microscope and are passed back through the optical system of the scanning microscope, either through the scanning optics to a dichroic mirror which reflects light at longer wavelengths while passing the shorter wavelength fluorescent light to a detector, or, by bypassing the scanning system and directing the light from the microscope objective lens to a dichroic mirror which passes the shorter wavelength fluorescent light directly to a detector. Such two photon systems are described in, e.g., Winfried Denk, et. al., xe2x80x9cTwo Photon Laser Scanning Fluorescence Microscopy,xe2x80x9d Science, Vol. 248, Apr. 6, 1990, pp. 73-76; Winfried Denk, et al., xe2x80x9cTwo-Photon Molecular Excitation in Laser-Scanning Microscopy,xe2x80x9d Chapter 28, Handbook of Biological Confocal Microscopy, Plenum Press, New York, 1995, pp. 445-448. If the incident light from the objective lens is focused to a narrow spot or waist in a semi-transparent specimen such that the intensity of the incident light is sufficient to excite multi-photon excitation only at the focal spot within the specimen, multi-photon fluorescence excitation will occur generally only in the focal plane. The fluorescent light emitted by the specimen can then be passed back and detected to obtain an image corresponding only to the focal plane and not to structures above and below the focal plane.
The laser light sources that are utilized to provide two (or more) photon excitation are generally selected to provide very short pulses of laser light, with a typical pulse width from a few picoseconds to several hundred femptoseconds. At such narrow pulse widths, the pulse modulation of the relatively long wavelength (substantially monochromatic) laser light effectively introduces higher and lower frequency components or sidebands in the pulse modulated light beam. The occurrence of such spectral spread may also be explained based on the uncertainty principle, which predicts that the shorter the temporal extent of the pulse, the wider its spectral content, regardless of how the pulse is produced. Thus, the spectral content of, e.g., a femptosecond (10xe2x88x9215 second) pulse is significantly larger than that of a picosecond (10xe2x88x9212 second) pulse. When beams with such narrow pulse widths are passed through an acousto-optic deflector (AOD), a diffractive element, the various wavelengths within the pulse modulated light tend to be spatially separated from one another by the AOD element. Such spatial dispersion of the pulses reduces the quality of the fluorescence image that can be obtained from such scanning systems.
In accordance with the present invention, a laser scanning microscope system utilizing short pulsed laser sources incorporates an acousto-optical deflector with compensation for spatial dispersion introduced by the deflector. In accordance with the invention, spatial dispersion of the pulses passed through the deflector is compensated by spatially compressing the pulses passed through the deflector to provide a spatially recombined pulse to the microscope optics.
In a laser scanning microscope system in accordance with the invention, pulsed laser light from a source is provided on an optical path defined by optical elements to the objective lens of microscope which focuses the laser light onto a specimen. The optical elements of the optical path include an acousto-optical deflector (or other chromatically dispersive scanning element) for deflecting the laser light selectively in one of the X or Y directions with respect to the specimen and a second deflector, such as a galvanometer driven mirror, to deflect the light in the other direction. Multi-photon fluorescent light emissions from the specimen are collected and directed to a fluorescent light detector, such as by collecting fluorescent photons incident on the objective lens and passing the fluorescent light back along the optical path to a light separation element such as a dichroic mirror which separates the fluorescent light from the reflected excitation light and directs the fluorescent light to a detector. Fluorescent light emitted in other directions, e.g., transmitted through the sample away from the objective lens, may be collected and detected. If desired, the system may also include elements for conventional laser scanning microscopy, such as a polarizing beam splitter for separating the reflected light at the excitation wavelength from the incoming laser light and directing such reflected light to a detector.
In the present invention, a dispersive prism is mounted adjacent to the output face of the acousto-optical detector to spatially compress the pulse which has been angularly spread by the acousto-optical deflector because of the spectral content of the pulse. The prism is preferably constructed so that the maximum correction is obtained at the center of the scanned angle. The beam exiting from the prism is directed to optical elements, such as lenses, which receive the output beam from the prism and direct it along the optical path to the objective lens of the microscope.
The optical path also preferably includes a mirror at the input side of the acousto-optical deflector which receives the beam on the optical path from the source and reflects it to the deflector. The relative angle between the mirror and the input face of the deflector are adjustable by mounting the mirror or the acousto-optical deflector, or both, to allow adjustment of the angular position between the face of the mirror and the input face of the acousto-optical deflector. The input angle to the acousto-optical deflector is preferably adjusted by appropriate adjustment of the angles between the mirror and the deflector so that the maximum diffraction efficiency (Bragg condition) is met at the desired wavelength of operation of the laser. However, the side wavelengths in the spectrum of the pulses will be somewhat Bragg-mismatched and thus attenuated. In this manner, the acousto-optical deflector also effectively acts as a spatial notch filter while allowing for the Bragg-matching of the center wavelength of the pulse. Utilization of an adjustable angle mirror combined with the input face of the acousto-optical deflector, and a dispersive prism mounted adjacent to the output face of the acousto-optical deflector, provide spatial compensation of the pulses with a minimum number of refractive elements and in a highly efficient and effective manner.
Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.